The genome sequence of the Purple Clay moth, Diarsia brunnea (Denis & Schiffermüller) 1775

We present a genome assembly from an individual male Diarsia brunnea (the Purple Clay moth; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence has a total length of 586.80 megabases. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.29 kilobases in length. Gene annotation of this assembly on Ensembl identified 18,730 protein-coding genes.


Background
The Purple Clay, Diarsia brunnea, is a macromoth of the Noctuidae (Noctuid) family, found throughout Britain and Ireland and across Europe and Asia as far as China and Japan (GBIF Secretariat, 2024).
In the UK its habitat is varied and includes broadleaved and mixed woodlands, moors, scrubby heathland, hedgerows and gardens.It is on the wing from late June to August.The Purple Clay is described as common, but not abundant, with its numbers probably declining though its distribution has not decreased (Randle et al., 2019) It is an attractive moth and the dry description given in modern textbooks does not give it the justice that a late nineteenth Lepidopterist does, who writes of the Purple Clay as 'beau-tiful……with its rich purple and red-brown ground colour, ochreous marblings and pale ochreous-grey reniform' (Tutt, 1896).The vernacular name 'Purple' refers to the purple tinge so evident in a fresh specimen, whereas its scientific name 'brunnea' refers to the brown undertone.
The Purple Clay hibernates as a caterpillar.The larvae feed by night retiring to leaf litter during the day.The diet of the larvae is truly catholic with a wide range of herbaceous plants in the autumn and in the spring more woody plants and even bracken.
The specimen was collected from a mercury vapour moth trap set up overnight on 16 June 2022 in Little Sparta, a garden designed by the artist Ian Hamilton Finlay in the 1960s, in South Lanarkshire, Scotland, surrounded by moorland and sheep farms.This species was identified by Jo Davis, an amateur lepidopterist and member of Butterfly Conservation.

Genome sequence report
The genome of an adult Diarsia brunnea (Figure 1) was sequenced using Pacific Biosciences single-molecule HiFi long reads, generating a total of 21.90 Gb (gigabases) from 2.13 million reads, providing approximately 36-fold coverage.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data, which produced 122.07 Gbp from 808.42 million reads, yielding an approximate coverage of 208-fold.Specimen and sequencing information is summarised in Table 1.
Manual assembly curation corrected three missing joins or mis-joins and one haplotypic duplications, increasing the scaffold number by 1.92%.The final assembly has a total length of 586.80 Mb in 52 sequence scaffolds with a scaffold N50 of 20.3 Mb (Table 2).The snail plot in Figure 2 provides a summary of the assembly statistics, while Figure 3 shows the base coverage against position per chromosome of the assembly, with the sequences coloured by phylum.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (99.72%) of the assembly sequence was assigned to 31 chromosomal-level scaffolds, representing 30 autosomes and the Z sex chromosome.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 3).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.

Sample acquisition and nucleic acid extraction
An adult Diarsia brunnea (specimen ID SAN00002568, ToLID ilDiaBrun1) was collected from Little Sparta, Dunsyre, Pentland Hills, Scotland, UK (latitude 55.72, longitude -3.51) on 2022-06-17 using a mercury vapour moth trap.The specimen was collected and identified by Jo Davis (Butterfly Conservation) and then preserved by flash freezing.
The specimen used for Hi-C and RNA sequencing (specimen ID SAN00002599, ToLID ilDiaBrun2) was collected from Isle of Bute, Mount Stuart, Scotland, UK (latitude 55.8, longitude -5.03) on 2022-07-02.The specimen was collected and identified by Dougie Menzies (Bute Natural History Society) and preserved on dry ice.
The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up.In sample preparation, the ilDiaBrun1 sample was weighed and dissected on dry ice (Jay et al., 2023).Tissue from the thorax was homogenised using a PowerMasher II tissue disruptor (Denton et al., 2023a).
HMW DNA was extracted in the WSI Scientific Operations core using the Automated MagAttract v2 protocol (Oatley et al., 2023).The DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system (Bates et al., 2023).Sheared DNA was purified by solid-phase reversible immobilisation (Strickland et al., 2023): in brief, the method employs AMPure PB beads to eliminate shorter fragments and concentrate the DNA.The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit dsDNA High Sensitivity Assay kit.Fragment size distribution was evaluated by running the sample on the FemtoPulse system.
RNA was extracted from abdomen tissue of ilDiaBrun2 in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mirVana protocol (do Amaral et al., 2023).The RNA concentration was assessed using a Nanodrop spectrophotometer and a Qubit Fluorometer using the Qubit RNA Broad-Range Assay kit.Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.
Protocols developed by the WSI Tree of Life laboratory are publicly available on protocols.io(Denton et al., 2023b).

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit.DNA and RNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences Sequel IIe (HiFi) and Illumina NovaSeq X (RNA-Seq) instruments.Hi-C data were also generated from head tissue of ilDiaBrun2 using the Arima-HiC v2 kit.The Hi-C sequencing was performed using pairedend sequencing with a read length of 150 bp on the Illumina NovaSeq 6000 instrument.
The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2023), which runs MitoFinder (Allio et al., 2020) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.

Assembly curation
The assembly was decontaminated using the Assembly Screen for Cobionts and Contaminants (ASCC) pipeline (article in preparation).Manual curation was primarily conducted using PretextView (Harry, 2022), with additional insights provided by JBrowse2 (Diesh et al., 2023) and HiGlass

Evaluation of the final assembly
A Hi-C map for the final assembly was produced using bwa-mem2 (Vasimuddin et al., 2019) in the Cooler file format (Abdennur & Mirny, 2020).To assess the assembly metrics, the k-mer completeness and QV consensus quality values were calculated in Merqury (Rhie et al., 2020).This work was Table 4 contains a list of relevant software tool versions and sources.

Genome annotation
The BRAKER2 pipeline (Brůna et al., 2021) was used in the default protein mode to generate annotation for the Diarsia brunnea assembly (GCA_949774965.1) in Ensembl Rapid Release at the EBI.

Wellcome Sanger Institute -Legal and Governance
The materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner.The Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.
The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material • Legality of collection, transfer and use (national and international)

Software tool Version
The data note titled "The genome sequence of the Purple Clay moth, Diarsia brunnea (Denis & Schiffermüller) 1775" presents the genome assembly of the Purple Clay moth.This moth has a wide distribution range across Europe and Asia.This is a good study with impact as a potential resource for future genomic studies, including population genetics and phylogeography of the moth.The methodology describing the genome assembly and annotation is sound, and the results present a comprehensive view of the study genome.The background of the insect is interesting; however, a minor addition would be a few lines to justify the study of the nuclear genome of the moth.

Is the rationale for creating the dataset(s) clearly described? Partly
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Molecular biology and Bioinformatics
We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.Reviewer Expertise: Omics technologies I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Figure 2 .
Figure 2. Genome assembly of Diarsia brunnea, ilDiaBrun1.1:metrics.The BlobToolKit snail plot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 586,790,279 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (36,429,188 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (20,336,188 and 13,737,000 bp), respectively.The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the lepidoptera_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilDiaBrun1_1/dataset/ilDiaBrun1_1/snail.

Figure 3 .
Figure 3. Genome assembly of Diarsia brunnea, ilDiaBrun1.1:Distribution plot of base coverage in ERR11029701 against position for sequences in assembly ilDiaBrun1_1.Windows of 100kb are coloured by phylum.The assembly has been filtered to exclude sequences with length < 2,550,000.An interactive version of this figure is available here.

Figure 4 .
Figure 4. Genome assembly of Diarsia brunnea ilDiaBrun1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all sequences.Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilDiaBrun1_1/dataset/ilDiaBrun1_1/cumulative.

Figure 5 .
Figure 5. Genome assembly of Diarsia brunnea ilDiaBrun1.1:Hi-C contact map of the ilDiaBrun1.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=CCsDWw10Qt-VDVvAJvS_-w.

Reviewer Report 06
September 2024 https://doi.org/10.21956/wellcomeopenres.25181.r95727© 2024 Walker A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Andrew A Walker University of Queensland, Queensland, Australia This report is of a high-quality nuclear genome sequencing and assembly of the lepidopteran Diarsia brunnea.It has been clearly prepared and I don't have specific suggestions to change it.I am not super familiar with what Wellcome Open Research requires in an article.This indexing just describes the sequencing and assembly of the genome, and does not analyse it further in a way relevant to lepidopteran biology.However I get the vibe that it is part of a larger project to enlarge genomic resources across the tree of life in general.Perhaps this could be clarified in the Background with a very minor change.Is the rationale for creating the dataset(s) clearly described?Partly Are the protocols appropriate and is the work technically sound?Yes Are sufficient details of methods and materials provided to allow replication by others?Yes Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.